Centrifuge having an improved centrifuge cell



24, 1965 K. STROHMAIER 3,202,348

GENTRIFUGE HAVING AN IMPROVED CENTRIFUGE CELL Filed Dec. 5. 1962 3 Sheets-Sheet 1 Fig.1

Y q/L-VIIIHIIIIIIIIA Aug. 24, 1965 K. STROHMAIER 3,262,348

CENTRIFUGE HAVING AN IMPROVED CENTRIFUGE CELL Filed Dec. 5, 1962 3 Sheets-Sheet 3 United States Patent f 3,202,348 'CEN'IRIFUGE HAVING AN IMPROVED CENTRIFUGE CELL Karl Strohmaier, Tubingen, Germany, assignor to Martin Christ, Osterode (Harz), Gipsmuhlenweg, Germany, a

corporation of Germany Filed Dec. 5, 1962, Ser. No. 242,492 Claims. (Cl. 23326) This invention relates to centrifuges, and more particularly to improved centrifuge cells adapted preferably to analytically determine the sedimentation constant of macromolecular dispersed substance, such as proteins, ferments, viri and the like. By sedimentation constant there is meant the migration velocity per centrifugal acceleration of the particles of the substance with respect to the liquid in which the particles are dispersed, which liquid is resting in the centrifugal field. This constant in general is determined by detecting the sedimentation, i.e. the settling of the particles in layers during rotation of the cell, by the changes occurring in the optical characteristics of the cell filling, and recording it. For determining the sedimentation constant, one preferably employs ultracentrifuges which due to the specific construction of the support arrangement, cushioning and drive mechanism and the presence of cooling means lend themselves to op: eration at very high speeds, preferably in the range above 10,000 r.p.m. Creation of turbulence in the cell filling during the centrifuging operation due to the influence of the inner walls of the cell can be prevented by giving those walls a sect-orial configuration.

When the concentration of the substance the sedimentation constant of which is to be determined is low, or when several substances having different sedimentation constants are simultaneously present in the liquid, the optical characteristics will change too little, or not sufiiciently distinctly, to permit the sedimentation constant of the dispersed substance or substances to be determined from such change. In such a case it is common practice to measure the change of the concentration of the fraction or fractions which are supernatant, and to compare the measured values with the starting material. In order to obtain exact results, it is required, however, that both tilting of the liquid or whirling up of the sediment layers during deceleration and stoppage be avoided and mixing of individual fractions during discharge thereof be prevented. While mixing and whirling up upon deceleration and during standstill can be prevented bynet-lilre partition walls or inserts and by the use of swinging-out cups for the cells, it heretofore was necessary that the various fractions of the sediment be withdrawn after centrifugation from the top of the cell by means of a small tube of appropriate shape, or the bottom of the cell be pierced with a small tube or cannula to permit the contents to drop out. Mixing of the fractions, however, upon immersion of the small tube or due to afterflow of liquid along the Walls could not be prevented in this Way.

Accordingly, it is an object of this invention to provide a centrifuge having a centrifuge cell which permits fractions to be Withdrawn without intermixing from the sedimentation zone of the cell.

Another object of this invention is to provide a centrifuge cell which in its wall has a plurality of channels which open at different levels and at the bottom of the sedimentation zone to permit withdrawal of fractions without intermixing.

3,292,348 Fatented Aug. 2%, 1965 Another object of this invention is to provide a centrifuge cell having its sedimentation zone constructed so that in the centrifugal field the sedimentation will take place radially without disturbance and without creation of turbulence.

Another object of this invention is to provide a centrifuge cell which between the sedimentation Zone and the open end of the cell has a space adapted to receive a device for pouring liquid over another without mixing.

Another object of this invention is to provide a centrifuge cell adapted to be inserted into cups which rotate about a vertical axis of rotation and swing out horizontally under the action of centrifugal acceleration and contain an immersion liquid surrounding the cell, the cell being constructed so as to effectively prevent leakage of immersion liquid thereinto.

Still another object of this invention is to provide a centrifuge cell adapted to be used in cups which rotate about a vertical axis of rotation and swing out horizontally under centrifugal acceleration, and the inside space of which is laterally bounded by a pair of substantially parallel wall surfaces and a pair of Wall surfaces extending perpendicularly thereto, said latter wall surfaces being disposed in planes intersecting in the axis of rotation when the cups are swung out completely, the side walls of said cups having provided therein a plurality of channels which open with one end into the unclosed front end and with their other end respectively at different levels and at the bottom of said inside space.

A still further object of the present invention is to a provide a double-chamber-type centrifuge cell from which the individual fractions can be withdrawn without any residues from the upper fractions being carried into lower fractions due to flow-on of liquid along the walls.

Other objects and advantages of this invention will become apparent from the following detailed description taken in conjunction with the attached drawings which, by way of example only, illustrate several embodiments of this invention.

In the drawings:

FIGURE 1 is a sectional view of a centrifuge including three outswinging cups each adapted to receive a cell embodying this invention,

FIGURE 2 is a longitudinal sectional view of trifuge cell embodying this invention,

FIGURE 3 is another longitudinal sectional view of the centrifuge cell shown in FIGURE 2,

FIGURE 4 is a sectional view taken along the line IV-IV of FIGURE 2.

FIGURE 5 shows a cover member adapted to be positioned onto the cell of this invention to seal the interior of the cell against the immersion liquid,

FIGURE 6 is a cross-sectional view of another em bodiment of the cell of this invention having planar surface sections on the outer Wall and channel opening ends which are narrowed in direction axially of the cell,

FIGURE 7 is a cross-sectional view of a device for pouring liquid over the contents of the cell withoutmixs,

FIGURE 8 is a sectional view of a portion of the device shOWn in FIGURE 7,

FIGURE 9 is a plan view of the device shown in FIG- URE 7,

FIGURE 10 is a cross-sectional view of a cell according to this invention with partition wall in condition prior to centrifuging, and

into horizontal position.

of the cup.

to a vacuum pump (not shown) adapted to evacuate chamber 4. A rotor 8 is mounted on the upper end of shaft 1 for rotation therewith.

In rotator 8 three centrifuge cups 9 are suspended by means of detachable thin horizontal axles 10 and equally spaced about the periphery of the rotor. In the drawing, only two such cups are shown, of which the left one is shown in its position at rest, while the right one is shown likewise at rest in dash-dot line at 912 and in swung-out position in solid line as well as in section.

When the rotor starts and runs up, cups 9 swing out At higher speeds the thin axles 10 will be slightly bent by the centrifugal force exerted thereon, so that the toroidal edges 11 of the cups 9 seat in and engage corresponding recesses 12 in the rotor.

Each cup 9 receives a centrifuge cell 13 and is closed by a cover 14 which, for example, has a screw thread and is screwed into a corresponding threaded portion Into the space formed between the inner Walls of cup 9 and the outer surfaces of the cell 13 there is preferably filled an immersion liquid which supports the walls of the cell against the pressure of the liquid in the cell created during centrifugation.

In the embodiment shown in FIGURES 2, 3, and 4, the centrifuge cell 13 of this invention is bounded at the bottom by a hemispherical outer surface 1-6 and laterally by a cylindrical outer surface 15. Near the open upper end of the cell, this outer surface 15 merges, preferably through a shoulder 26, in a cylindrical outer surface 17 of reduced diameter, whereby, when an immersion liquid is present between cell 13 and cup 9, an annular free space is provided, which prevents this liquid from penetrating into the interior of the cell.

As an alternative, however, the cylindrical outer surface 15 may also extend, without reduction in diameter, up to the open upper end, in which case leakage of immersion liquid is prevented by a cap 18 (FIGURE 5). Such a cell may have, for example, an outer axial length of about 75 mm. and an outer diameter of about 24 mm.

The sedimentation zone 19 of the cell is bounded by four plane wall surfaces 20, 21 and 22, 23 disposed vertically to each other and a conical bottom surface 24. Wall surfaces 20, 21 are parallel to each other and extend from bottom surface 24 through the sedimentation zone 19 to a shoulder 26 at the lower end of an overpouring zone 25 to be described in more detail hereinbelow. The two opposed wall surfaces 22, 23 extend from the bottom surface 24 to a shoulder 26 adjoining the overpouring zone 25. Wall surfaces 22, 23 are located in planes which, when the cup is in position fully swung out, intersect in the axis of rotation of shaft 1 of the centrifuge, that is to say wall surfaces 20, 21 are disposed in substantially horizontal planes when the apparatus is in operation.

If for a given diameter of the cup and a predetermined wall thickness of the cell, the sedimentation space 19 of 1 cell 13 is to have a maximum volume, the sedimentation space must be of square cross section in a plane vertical to the longitudinal axis and closely above the bottom of the cell; that is, the inner surfaces 20, 21, 22 and 23 must be of equal width at their lower ends.

The overpouring space 25 is of cylindrical configuration and extends from shoulder 26 to the open front end of the cell, and it is adapted to receive means for pouring liquid over the contents of the cell without mixing,

A l which will be described hereinafter, by way of example, with reference to FIGURES 7 through 9.

In the walls of the cell there are formed between the inner and outer surfaces a plurality of channels or passageways 27 which preferably are of circular cross section and open with their one ends at the open front end of the cell and with their respective other ends into the sedimentation zone 19 at dilferent heights or levels at 28 and at the bottom at 29, respectively.

In a preferred embodiment, the passageways have cylindrically enlarged portions 36 at their respective orifices at the front or open end of the cell. The orifices 28 at the ends of the passageways leading from the sides into the sedimentation zone, to advantage, are very narrow in axial direction of the cell while being wide in direction vertical thereto so as to define slots 31 which occupy substantially the entire width of an inner wall surface (FIGURE 6). In cases where the cell is to be used preferably for optical observation, two outer surfaces 40 are provided that are arranged in parallel relation to each other and to the parallel inner surfaces.

The cell preferably is made in one piece and of a material which preferably is transparent, such as glass or polyacrylate ester.

The sedimentation constant of macromolecular dispersed matter can be determined by means of a cell constructed in accordance with this invention e.g. by first filling the cell, prior to centrifugation, with a pure liquid (gradient) up to a predetermined level. For the purpose of stabilization, a low density gradient is created in this filled-in liquid column by a different content of sugar or heavy water or some other substance which increases the density of the liquid. Then the solution containing the particles to be measured is poured, without mixing, over this liquid column in a narrow zone, it being required in this connection that the density of the solution poured over be lower than the lowest density of the subjacent liquid layer.

Such overpouring without mixing can be effected prior to the centrifugation, or during running up of the centrifuge, by means of apparatus positioned in the overpouring zone 25. During centrifugation itself, however, this pouring over must be terminated and any convection must be strictly avoided.

A suitable device for pouring over without mixing is illustrated by way of example, in FIGURES 7 through 9. The device comprises an outer tubular member 32 which at its underside includes a conical portion 33 having a central aperture 34 therein and connected to an inner tubular member 35 preferably of shorter length. The dimensions of the outer member 32 are selected so that the overpouring device can be positioned within the overpouring space 25 and rests on shoulder 26 during operation. Near the upper end of outer member 32, one or more, preferably diagonally opposed, bores 36 having a diameter of eg 1 mm. are provided to permit removal of the overpouring device from the cell, to which end one preferably uses a hairpin-shaped hook, not shown, having its ends bent outwardly at right angles. Furthermore, one or more narrow bores 37 of e.g. 0.3 mm. diameter are provided near the lower end of outer member 32 and open inside closely above the conically shaped bottom 33 or extend a short distance further through the bottom as an open top short channel 38.

Before starting with the test, the narrow bores 37 are stopped up e.g. with Vaseline and, after the gradient has been filled in the cell, the device is placed within the cylindrical overpouring space 25. Thereafter the anular space 39 is filled with the solution to be poured over without; mixing. During running up of the centrifuge, when a: sufiiciently high speed is reached, the Vaseline will be: forced by the solution out from the bores 37, whereupon.

the solution is free to flow along the wall onto the sur-- face of the gradient. Owing to the density difference,

which is further raised by the action of centrifugal force,

the solution will be layered neatly over the gradient.

Through the central aperture 34 in the overpouring device there can pass a beam of light rays which, when the concentration is sufficiently high, permits also an optical observation of the position of a macromolecular substance which is light-dispersing or detectable in any other Way.

After the centrifugation has been completed, the contents of the cell can be sucked out from the sedimentation zone 19 through the passageways 27 in the individual unmixed fractions by attaching to the end openings of the passageways the cannula. of a syringe or some other suitable suction device. By means of biological, chemical or physical measurements, it can be determined to which depth the particles to be studied have migrated during the period of centrifuging. The sedimentation constant can then be calculated from the level difference of the active zone after centrifugation and the position of the poured-over zone before centrifugation and from the centrifugation time and the acceleration that has become effective.

With the passageway opening at 29 at the bottom, the liquid column in the sedimentation zone 19 can be raised or lowered so as to present a'particular desired fraction before a particular channel opening 28. When suction is made through a particular channel 27, the liquid fraction above the level of channel opening 28 will be drawn off, whereby this fraction slides on the liquid below the level of said opening into said opening. It may occur that during such sliding some of the liquid diffuses from the fraction to be drawn off into the subjacent layer. If one thereupon were to draw off the subjacent fraction over the volume between the above-mentioned channel opening and the next following opening therebel'ow, liquid from the upper fraction or fractions might be carried off through the mixing zone.

In accordance with the present invention, this can be effectively prevented by employing two equally treated cells in which the channel openings are offset with respect to each other. In that case, the lower half of a particular faction is withdrawn from the first cell, while the upper half of: the same fraction is withdrawn from the second cell which has offset its channel openings 28 by one half of the level difference between the respective openings in the first cell. In this way, respectively the upper and lower halves of the fractions, and thus the boundary or mixing zones will remain in each cell and are not utilized for the test.

In accordance with this invention, there is provided another embodiment of the cell in the form 'of a doublechamber cell, which eliminates the need for two separate cells, and yet prevents in the same manner, as described above, the withdrawal of fractions having portions of other fractions diffused thereinto. While, where two cells are employed, special care must be taken to ensure that exactly equal quantities of the suspension or liquid to be tested are poured-over at the beginning of the centrifugation to preclude any errors in the evaluation of the measurement results, this requirement will be automatically set in the double-chamber cell of this invention. Such a double-chamber cell indicated generally at 41 is schematically shown, in longitudinal section, in FIGURE 10.

This cell substantially has the same external configuration as described in conjunction with FIGURES 2 and 3. The sedimentation space 19 likewise is bounded by four relatively vertically extensive wall surfaces, two of which are disposed parallel to each other and to the plane of the drawing, and the other two (22, 23) are located in planes intersecting substantially in the axis of rotation of the centrifuge shaft when the cup receiving the cell is in position fully swung out. As an alternative, the wall surfaces may also be constructed e.g. so that the sedimentation space 19 will be of cylindrical, conical or prismshaped configuration, just as in the previously described embodiments.

The sedimentation space 19 is divided into two chambers 43 and 44, preferably of equal size, by a partition wall 42 formed integrally with the cell and extending lengthwise thereof. Partition wall 42 preferably is disposed in a plane which includes the center axis of the centrifuge cups. In the embodiment shown in FIGURES 10 and 11 the plane of the wall 42 includes also the axis of rotation of the centrifuge. Alternatively, the partition wall 42 may also be formed of a material other than that of the cell, or may be oriented differently, such as vertically with respect to the orientation shown, or may be constructed so as to be removable. Partition wall 42 has a height lower than that of the sedimentation space 19 to leave a common space 45 above the two chambers 43 and 44.

From the front end of the cell, separate channels 27 extend downwardly to different levels of the sedimentation space. These channels or passageways 27, of which only the uppermost ones are shown, in the schematic representation, as extending through to the front end, for the sake of clarity, open alternately into the two chambers 43 and 44 and into the common space 45, respectively, and the openings preferably are equally spaced in direction axially of the cell, with one such opening being provided at the levelof the upper edge of partition wall 42. Alternatively, e.g. for the event that by the selection of the previously mentioned density gradient the traveling velocity of the particles varies inversely proportionately to the respective traveling velocities of the sedimentcd particles at different levels. It is not compulsory that, as shown in FIGURES 10 and 11, the openings of the passageways, or the passageways 27 themselves, are disposed in the same plane. Thus, for example, the passageways may also open in the parallel wall surfaces of the sedimentation space and/or may extend in inclined direction in the wall of the cell.

The operation of the above-described double-chamber cell is as follows: After a liquid having the same density gradient has been filled into chambers 4-3 and 44, the overlying liquid 46 is poured as a whole over both chambers 43 and 44, such as by means of the equipment shown in FIGURES 7 through 9. In this way, an exactly quantity and concentration of the substance to be tested will be present over each of the chambers 43 and 44.

In FIGURE 11, the position of the poured-over or overlying liquid 46 after centrifuging is indicated at 47, and a possibility of withdrawing the individual layers from the sedimentation space is shown.

First of all, the supernatant liquid in the space 45 above both chambers 43 and 44 is discharged through the upper, left (as viewed in the drawing) passageway. Then the various fractions 48 indicated by horizontal lines in the drawing are separately sucked off from top downward through the respective passageways. The liquid in the intermediate layers which also flows on downwardly, but is not discharged, effectively eliminates any possibility of overlying fractions being carried in undesirable manner into lower fractions. As the contents of both chambers 43 and 44 compulsorily have been treated in an entirely identical manner, the various individual fractions from the different chambers can be strictly compared respectively with each other. a

From the above description, it will become apparent that the cell of this invention may be used not only for determining the sedimentation constant but also for separating a solution in which several substances having different sedimentation constants or different densities are dissolved. Although the inventionhas been described in conjunction with a cell having a sector-shaped sedimentation space, it will be clear to those skilled in the art that modification and changes may be made both in the sedimentation space and the external configuration of the cell without departing from the spirit and scope of the appended claims.

Iclaim:

1. A centrifuge comprising a rotatable member, at least one cup pivotally connected suitably near its open end to said member to swing outwardly in response to centrifugal force as said member rotates, and a centrifuge cell in the form of an open-ended, closed bottom tube disposed with its closed bottom seated in said cup, said 'cup being free to swing outwardly until the longitudinal axis of said cell is approximately horizontal, and said cell having a plurality of passageways in its side walls extending lengthwise thereof, each passageway opening at one end through the open end of the cell, the other end of one passageway opening into the closed bottom of the cell and the other ends'of the other passageways opening into the interior of the cell, each at a different level.

2. The centrifuge of claim 1 in which each of the ends of the passageways opening into the interior of the cells at different levels is in the form of a slot extending in a direction transversely of the cell.

3. A centrifuge comprising a rotatable member, at least one cup pivotally connected suitably near its open end "to said member to swing outwardly in response to centrifugal force as said member rotates, said cup containing an immersion liquid, and a centrifuge cell in the form of an open-ended, closed bottom tube disposed with its closed bottom seated in said cup and being of reduced diameter in a region near its open end, said cup being free to swing outwardly until the longitudinal axis of said cell is approximately horizontal, and said cell having a plurality of passageways in its-side walls extending length- ,wise thereof, each passageway opening at one end through the open endof the cell, the other end of one passageway opening into the closed bottom of the cell and the other ends of the other passageways opening into the interior of the cell, each at a different level.

4. A centrifuge comprising a rotatable member, at least 'one cup pivotally connected suitably near its open end to said member, to swing outwardlyyin response to centrifugal force as said member rotates, and a centrifuge cell in the form of an open-ended, closed bottom tube disposed with its closed bottom seated in said cup, the interior space of said cell being laterally bound by a pair of substantially parallel wall surfaces and another pair of wall surfaces extending vertically with respect thereto and in planes intersecting in the axis of rotation of said member when the cup is fully swung out, said cup being free to swing outwardly until the longitudinal axis of said cell is approximately horizontal, and said cell having a plurality of passageways extending lengthwise in its side walls, each passageway opening at one end through the open end of the cell, the other end of one passageway opening into the closed bottom of the cell and the other ends of the other passageways opening into the interior of the cell each at a different level.

5. The centrifuge of claim 4 in which the interior of the cell is of square crosssection in the region closely above the bottom end thereof.

6. A centrifuge comprising a rotatable member, at least one cup pivotally connected suitably near its open end ,ended space and a sedimentation space, said sedimentation space bieng laterally bound by a pair of substantially parallel wall surfaces and another pair of wall surfaces extending vertically with respect thereto and in planes intersecting in the axis of rotation of said member when the cup is fully swung out, and establishing shoulders at the junction of said spaces, said cup being free to swing outwardly until the longitudinal axis of said cell is approximately horizontal, and said cell having a plurality of passageways extending lengthwise in its side walls, each passageway opening through the open end of the cell,

one passageway opening into the closed bottom of the cell and the other passageways opening into the interior of the cell each at a different level.

7. A centrifuge comprising a rotatable member, at least one cup pivotally connected suitably near its open end to said member to swing outwardly in response to centrifugal force as said member rotates, and a centrifuge cell in the form of an open-ended, closed bottom tube disposed with its closed bottom seated in said cup, said cup being free to swing outwardly until the 1ongitudinal axis of said cell is approximately horizontal, said cell having a plurality of passageways in its side Walls extending lengthwise thereof, each passageway opening at one end through the open end of the cell, the other end of one passageway opening into the closed bottom of the cell and the other ends of the other passageways opening into the interior of the cell each at a different level, and an overpouring device including an outer wall within the open end of the tube, a central tubular wall, and a bottom wall of conic section interconnecting the inner ends of said walls, said device having at least one outwardly opening bore extending through said outer wall near its junction with said bottom wall.

8. The centrifuge of claim 7 in which the cell has spaced shoulders supporting said bottom wall of said device.

9. A centrifuge comprising a rotatable member, at least one cup pivotally connected suitably near its open 'end to said member to swing outwardly in response to centrifugal force as saidmember rotates, and a centrifuge cell in the form of an open-ended, closed bottom tube of I transparent stock disposed with itsclosed bottom'seated in said cup, the interior space of said cell being laterally bound by a pair of substantially parallel wall surfaces and another pair of wall surfaces extending vertically with respect thereto and in planes intersecting in the axis of rotation of said member when the cup is fully swung out, said cup being free to swing outwardly until the longitudinal axis of said cell is approximately horizontal, and said cell having a plurality of passageways extending lengthwise in its side walls, each passageway opening through the open end of the cell, onepassageway opening into the closed bottom of the cell and the other passageways opening into the interior of the cell each at a different level, said tube having a generally cylindrical outer surface which has at least one'plane area parallel 'to said first named wall surfaces.

10. A centrifuge comprising a rotatable member, at least one cup pivotally connected suitably near its open end to said member to swing outwardly in response to centrifugal force as said member rotates, and a centrifuge cell in the form of an open-ended, closed bottom tube disposed with its closed bottom seated in saidcup, said cup being free to swing outwardly until the longitudinal axis of said cell is approximately horizontal, a partition extending upwardly from the bottom a substantial distance but terminating short of the open cell end and dividing the interior of the cell into equal chambers, and said cell having a plurality of passageways in its side wallsextending lengthwise thereof,each passageway opening at one end through the open end of the cell, and the other ends of the passageways opening into the interior of the cell at a different level in a predetermined manner, some on one side of the partition and the others at the other side thereof.

References Cited by the Examiner UNITED STATES PATENTS 562,782 6/96 Hoyt 233-28 585,936 7/97 Linders 233-28 676,631 6/01 Ohlhaver 233-46 X 834,014 10/06 Lyke 215-1 J 974,075 10/10 King 233-46 X 1,196,880 9/16 Nickerson 210-537 X- (Dtherreferences on following page) 9 UNITED STATES PATENTS Galleguillos 215-1 Rubissow 233-26 Williams et a1. 23259 X Huenergardt 128--272 X 5 Whitby 23326 X Anderson 233-26 Danielsson et a1 233-26 Deaver 233-26 X FOREIGN PATENTS 480,887 7/16 France. 299,909 8/17 Germany.

OTHER REFERENCES German printed application 1,094,488, December 12, 1960.

HARRY B. THORNTON, Primary Examiner.

Gauslaa 233---26 10 ROBERT F. BURNETT, Examiner. 

1. A CENTRIFUGE COMPRISING A ROTATABLE MEMBER, AT LEAST ONE CUP PIVOTALLY CONNECTED SUITABLY NEAR ITS OPEN END TO SAID MEMBER TO SWING OUTWARDLY IN RESPONSE TO CENTRIFUGAL FORCE AS SAID MEMBER ROTATES, A CENTRIFUGE CELL IN THE FORM OF AN OPEN-ENDED, CLOSED BOTTOM TUBE DISPOSED WITH ITS CLOSED BOTTOM SEATED IN SAID CUP, SAID CUP BEING FREE TO SWING OUTWARDLY UNTIL THE LONGITUDINAL AXIS OF SAID CELL IS APPROXIMATELY HORIZONTAL, AND SAID CELL HAVING A PLURALITY OF PASSAGEWAYS IN ITS SIDE WALLS EXTENDING LENGTHWISE THEREOF, EACH PASSAGEWAY OPENING AT ONE END THROUGH THE OPEN END OF THE CELL, THE OTHER END OF ONE PASSAGEWAY OPENING INTO THE CLOSED BOTTOM OF THE CELL AND THE OTHER ENDS OF THE OTHER PASSAGEWAYS OPENING INTO THE INTERIOR OF THE CELL, EACH AT A DIFFERENT LEVEL. 